Immunogenicity is the ability to, or the degree to which a particular substance may provoke an immune response, such as the production of antibodies. In immunogenicity studies, there is an increasing interest to measure and quantify the components “hidden” in immune complexes. For example, in the study of protein drugs, such as therapeutic antibodies, it is of interest to detect antibodies elicited against the drug. However, since the drug may be present in rather high concentrations in sera, the anti-drug antibodies may form immune complexes with the drug to a large extent. In the case of immunotherapeutic proteins used to elicit an immune reaction against a specific disease-causing protein, the situation is the opposite. The antibodies are in a great excess relative to the antigen, and it is of interest to know how much antigen that is complex-bound, since high levels of immune complexes may start complex activation. There are, however, today no convenient and efficient techniques for measuring the total concentration of free and complex-bound analyte in a serum sample, or how much analyte that is in free and complex form, respectively.
Moxness, M. S., et al., “Immunogenicity Testing for Antibodies Directed Against Therapeutic Human Monoclonal Antibodies Using Electrochemiluminescent Detection”, Abstract 59 and Poster, 37th Annual Oak Ridge Conference, April 14 & 15, 2005, Baltimore, Md., disclose an assay for monitoring immune response against human therapeutic monoclonal antibodies (drugs). A rabbit polyclonal antibody specific for each drug was used as surrogate analyte and added to serum. Drug was then added, and the serum was treated with acid (pH 3.3) for one hour to dissociate analyte-drug complexes, brought to neutral pH and assayed. In the assay, the neutralized serum sample was incubated over night with (i) drug conjugated with a ruthenium complex that emits light through electroluminescence (ECL), and (ii) drug conjugated with biotin. The mixture was then transferred to streptavidin-coated plates equipped with electrodes to capture biotin-drug/analyte-ruthenium drug complexes, and ECL signals were measured on an ECL-analyzer and normalized against a negative control in every assay. However, all complex-bound analytes can not be measured since analyte complexed with biotin-drug conjugate at both binding sites, or ruthenium-drug conjugate at both binding sites will not be detected, and a determination of the total analyte concentration can therefore not be obtained.
Tomimori-Yamashita, J., et al., Lepr. Rev. (1999) 70: 261-271 discloses determination of anti-PGL-I specific circulating immune complex in leprosy patients. The circulating immune complexes in sera were precipitated by adding polyethylene glycol and isolate the precipitate by centrifugation. After dissolving the precipitate in EDTA, the solution was acidified with HCl-glycine and then neutralized with potassium hydrogenphosphate. The levels of IgG or IgM antibodies against PGL-I were then tested by ELISA within 30 minutes by incubating the solution in PGL-I coated wells for 90 to 180 minutes, reacting with enzyme conjugate and substrate, and spectrophotometrically reading the color developed through the enzyme activity. However, as is well known in the art, PEG does not precipitate all complexed analytes, and a determination of the total analyte concentration will therefore not be obtained
Both the above described prior art methods require lengthy incubations and are therefore not suited for flow cell assay formats. There is therefore a need for a quick and easy-to-perform assay that permits determination of free as well as any complexed analyte, which is capable of detecting all complexed analytes and which also is well suited for flow cell applications.